Bacterial infections and the resulting burden on society and health care management around the globe remains a tremendous challenge. Vaccines against important bacterial diseases such as MenACWY, MenB, group A Streptococcal (GAS) or Group B Streptococcal (GBS) disease, Streptococcus pneumoniae, Pseudomonas aeruginosa are currently in pre-clinical or clinical testing. The method currently employed by most laboratories for testing the efficacy of a vaccine involves testing clinical samples obtained from subjects inoculated with the vaccine for serum bactericidal activity (SBA) by counting surviving bacterial colony as a measure of the ability of a vaccine to induce a serum response in a vaccinated subject to effect killing of the bacterial pathogen tested. Functional assays such as a serum bactericidal assay are used as a proxy for efficacy based upon the assumption that if the subject has produced bactericidal antibodies against the pathogen above a specified level, then the subject is protected against infection by the organism and therefore that the vaccine may be used to protect others against the pathogen. These responses are measured in mice and humans and are a standard indicator of vaccine efficacy (e.g. see end-note 14 of reference (R5) below). Serum bactericidal activity measures bacterial killing mediated by complement, and can be assayed using human or animal complement, such as baby rabbit complement. WHO standards require a vaccine to induce at least a 4 fold rise in SBA in more than 90% of recipients when rabbit complement is used. Published studies (Goldschneider et al.) assigned an SBA titer of 1:4 using human complement as a correlate of protection against meningococcal disease. The functional assay and threshold as used today is typically an underestimate of a vaccine's efficacy, but such underestimate is deemed in the best interest of the public. Since the traditional SBA assay requires plating bacteria onto agar and counting surviving colony forming units (cfu) after an overnight incubation step, such assays are time consuming as the assays often take two or more days, are not simple to use and therefore problematic to standardize, and are labor-intensive resulting into low sample throughput. Thus the traditional SBA assays are not amenable to highly accurate and high-throughput rapid data analysis from clinical trials. In particular, due to the difficulty of assay standardization among labs, the generation of reliable data with large and multi-site clinical trials or from post-licensing surveillance can be problematic. Consequently there is an acute and long-felt need to develop a sensitive, high throughput bactericidal assay to enable a rapid and highly reproducible assessment of the efficacy of bacterial vaccine candidates.
The problems regarding the traditional SBA assay mentioned above have been recognized, but have not been solved. The usage of ALAMARBLUE™ (resazurin) has been initially described to measure the viability of cells exposed to poising agents (U.S. Pat. No. 5,501,959). Viable cells reduce ALAMARBLUE™ changing the color from blue to pink for colorimetric measurement and are emitting a fluorescence signal after excitation. For SBA assays the inclusion of ALAMARBLUE™ to measure fluorescence instead of counting surviving bacterial colonies was reported suggesting a good correlation between the traditional and the fluorescence based SBA assay (Mountzouros et al., 2000; Romero-Steiner et al., 2004).
However, until the present application the signal was either measured in plates using agar-containing medium (Mountzouros et al., 2000), which is unsuitable using automated liquid handlers, or only single end-point measurements were recorded (Romero-Steiner et al., 2004). Moreover, the reaction plates remained unsealed during the assay (Mountzouros et al., 2000; Romero-Steiner et al., 2004), increasing the risk of inconsistent results due to cross-contamination and evaporation. Thus, neither has to date been adapted into a commercially available high-throughput assay system to measure bactericidal activity, despite the long-felt and recognized need.